The biotrophic basideomycete fungus Puccinia psidii is the causal agent of rust in Eucalyptus spp. and other plants of the family Myrtaceae. The Eucalyptus rust has caused major economic and yield losses (up to 60% in young plants) to the pulp and paper industry in Brazil. Several molecular tools have been developed for the detection and monitoring of phytopathogens in their hosts, emphasizing the Polymerase Chain Reaction (PCR) in which the microorganism are indirectly detected by specific genetic fragments. Thus, the main goal of this work is to design and validate specific primers for P.psidii in order to detection and future monitoring this fungus during the infection process in Eucalyptus grandis susceptible genotypes, using a quantitative PCR (qPCR). To achieve this objective, DNA from uredospores of P. psidii (isolate MF-01) was extracted, the intergenic spacer region (IGS) of rDNA was sequenced and compared with sequences from other species of the genus Puccinia (Blast-n, NCBI database). We did similar procedure for development other two pairs of primers for other constitutive genes of P. psidii: the elongation factor (EF) and beta tubulin (BT). The three pairs of primers designed, were compared with the Eucalyptus genome database, to avoid non-specific amplification. To optimize and to validate the primers specificity, we did a conventional PCR with the DNA of E. grandis leaves infected and non-infected with P.psidii, the DNA of another basidiomycete Sporosorium scitamineum, and the DNA of P.psidii uredospores. The best found condition was used to the qPCR reactions. As expected by conventional PCR, fragments of 150-200bp were amplified only to P. psidii DNA and infected plant samples. Similar results were found using the qPCR, demonstrating that the designed primers are sensitive and specific to detection of P.psidii. Thus, in future, these set of primers may be used for monitoring by qPCR, this phytopathogen during the early stage infection process in E.grandis leaves, being an important tool to analysis of control of the fungus on eucalyptus plants.